Antisense oligonucleotides of human regulatory subunit RI-.sub.α of camp dependent protein kinases for the treatment of cancer

ABSTRACT

Antisense oligonucleotides of human regulatory subunit RI-alpha of cAMP-dependent protein kinases are disclosed along with pharmaceutical compositions containing these oligonucleotides as the active ingredients. These antisense oligonucleotides are useful for inhibiting the growth of cancer.

The present invention was made with government support. Accordingly, theUnited States government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of application Ser. No. 07/702,163, filedMay 20, 1991, now U.S. Pat. No. 5,271,941, which is acontinuation-in-part of U.S. application Ser. No. 07/680,198, abandoned,filed Apr. 5, 1991, which is a continuation-in-part of U.S. applicationSer. No. 07/607,113, filed Nov. 2, 1990 abandoned, the contents of whichare fully incorporated by reference herein.

FIELD OF THE INVENTION

The invention is in the field of medicinal chemistry. In particular, theinvention relates to certain antisense oligonucleotides and the usethereof for the treatment of cancer.

BACKGROUND OF THE INVENTION

Control mechanisms for cell growth and differentiation are disrupted inneoplastic cells (Potter, V. R. (1988) Adv. Oncol. 4, 1-8; Strife, A. &Clarkson, B. (1988) Semin. Hematol. 25, 1-19; Sachs, L. (1987) CancerRes. 47, 1981-1986). cAMP, an intracellular regulatory agent, has beenconsidered to have a role in the control of cell proliferation anddifferentiation (Pastan, I., Johnson, G. S. & Anderson, W. B. (1975)Ann. Rev. Biochem. 44, 491-522; Prasad, K. N. (1975) Biol. Rev. 50,129-165; Cho-Chung, Y. S. (1980) J. Cyclic Nucleotide Res. 6, 163-177;Puck, T. T. (1987) Somatic Cell Mot. Genet. 13, 451-457). Eitherinhibitory or stimulatory effects of cAMP on cell growth have beenreported previously in studies in which cAMP analogs such as N⁶ -O^(2')-dibutyryladenosine 3',5'-cyclic monophosphate or agents that raiseintracellular cAMP to abnormal and continuously high levels were used,and available data are interpreted very differently (Chapowski, F. J.,Kelly, L. A. & Butcher, R. W. (1975) Adv. Cyclic Nucleotide ProteinPhosphorylat. Res. 6, 245-338; Cho-Chung, Y. S. (1979) in Influence ofHormones on Tumor Development, eds. Kellen, J. A. & Hilf, R. (CRC, BocaRaton, Fla.), pp. 55-93); Prasad, K. N. (1981) in The Transformed Cell,eds. Cameron, L. L. & Pool, T. B. (Academic, New York), pp. 235-266;Boynton, A. L. & Whitfield, J. F. (1983) Adv. Cyclic Nucleotide Res. 15,193-294).

Recently, site-selective cAMP analogs were discovered which show apreference for binding to purified preparations of type II rather thantype I cAMP-dependent protein kinase in vitro (Robinson-Steiner, A. M. &Corbin, J. D. (1983) J. Biol. Chem. 258, 1032-1040; O/greid, D.,Ekanger, R., Suva, R. H., Miller, J. P., Sturm, P., Corbin, J. D. &Do/skeland, S. O. (1985) Eur. J. Biochem. 150, 219-227), provoke potentgrowth inhibition, differentiation, and reverse transformation in abroad spectrum of human and rodent cancer cell lines (Katsaros, D.,Tortora, G., Tagliaferri, P., Clair, T., Ally, S., Neckers, L., Robins,R. K. & Cho-Chung, Y. S. (1987) FEBS Lett. 223, 97-103; Tortora, G.,Tagliaferri, P., Clair, T., Colamonici, O., Neckers, L. M., Robins, R.K. & Cho-Chung, Y. S. (1988) Blood, 71, 230-233; Tagliaferri, P.,Katsaros, D., Clair, T., Robins, R. K. & Cho-Chung, Y. S. (1988) J.Biol. Chem. 263, 409-416). The type I and type II protein kinases aredistinguished by their regulatory subunits (RI and RII, respectively)(Corbin, J. D., Keely, S. L. & Park, C. R. (1975) J. Biol. Chem. 250,218-225; Hofmann, F., Beavo, J. A. & Krebs, E. G. (1975) J. Biol. Chem.250, 7795-7801). Four different regulatory subunits [RI.sub.α(previously designated RI) (Lee, D. C., Carmichael, D. F., Krebs, E. G.& McKnight, G. S. (1983) Proc. Natl. Acad. Sci. USA 80, 3608-3612),RI.sub.β (Clegg, C. H., Cadd, G. G. & McKnight, G. S. (1988) Proc. Natl.Acad. Sci. USA 85, 3703-3707), RII.sub.α (RII₅₄) (Scott, J. D., Glaccum,M. B., Zoller, M. J., Uhler, M. D., Hofmann, D. M., McKnight, G. S. &Krebs, E. G. (1987) Proc. Natl. Acad. Sci. USA 84, 5192-5196) andRII.sub.β (RII₅₁) (Jahnsen, T., Hedin, L., Kidd, V. J., Beattie, W. G.,Lohmann, S. M., Walter, U., Durica, J., Schulz, T. Z., Schlitz, E.,Browner, M., Lawrence, C. B., Goldman, D., Ratoosh, S. L. & Richards, J.S. (1986) J. Biol. Chem. 261, 12352-12361)] have now been identified atthe gene/mRNA level. Two different catalytic subunits [C.sub.α (Uhler,M. D., Carmichael, D. F., Lee, D. C. Chrivia, J. C., Krebs, E. G. &McKnight, G. S. (1986) Proc. Natl. Acad. Sci. USA 83, 1300-1304) andC.sub.β (Uhler, M. D., Chrivia, J. C. & McKnight, G. S. (1986) J. Biol.Chem. 261, 15360-15363; Showers, M. O. & Maurer, R. A. (1986) J. Biol.Chem. 261, 16288-16291)] have also been identified; however,preferential coexpression of either one of these catalytic subunits witheither the type I or type II protein kinase regulatory subunit has notbeen found (Showers, M. O. & Maurer, R. A. (1986) J. Biol. Chem, 261,16288-16291).

The growth inhibition by site-selective cAMP analogs parallels reductionin RI.sub.α with an increase in RII.sub.β, resulting in an increase ofthe RII.sub.β /RI.sub.α ratio in cancer cells (Ally, S., Tortora, G.,Clair, T., Grieco, D., Merlo, G., Katsaros, D., O/greid, D., Do/skeland,S. O., Jahnsen, T. & Cho-Chung, Y. S. (1988) Proc. Natl. Acad. Sci. USA85, 6319-6322; Cho-Chung, Y. S. (1989) J. Natl. Cancer Inst. 81,982-987).

Such selection modulation of RI.sub.α versus RII₆₂ is not mimicked bytreatment with N⁶,O^(2') -dibutyryladenosine 3',5'-cyclic monophosphate,a previously studied cAMP analog (Ally, S., Tortora, G., Clair, T.,Grieco, D., Merlo, G., Katsaros, D., O/greid , D., Do/skeland, S. O.,Jahnsen, T. & Cho-Chung, Y. S. (1988) Proc. Natl. Acad, Sci. USA 85,6319-6322). The growth inhibition further correlates with a rapidtranslocation of RII.sub.β to the nucleus and an increase in thetranscription of the RII₆₂ gene (Ally, S., Tortora, G., Clair, T.,Grieco, D., Merlo, G., Katsaros, D., O/greid , D., Do/skeland, S. O.,Jahnsen, T. & Cho-Chung, Y. S. (1988) Proc. Natl. Acad. Sci. USA 85,6319-6322). These results support the hypothesis that RII₆₂ plays animportant role in the cAMP growth regulatory function (Cho-Chung, Y. S.(1989) J. Natl. Cancer Inst. 81, 982-987).

Antisense RNA sequences have been described as naturally occurringbiological inhibitors of gene expression in both prokaryotes (Mizuno,T., Chou, M-Y, and Inouye, M. (1984), Proc. Natl. Acad. Sci. USA 81,(1966-1970)) and eukaryotes (Heywood, S. M. Nucleic Acids Res., 14,6771-6772 (1986)), and these sequences presumably function byhybridizing to complementary mRNA sequences, resulting in hybridizationarrest of translation (Paterson, B. M., Roberts, B. E., and Kuff, E. L.,(1977) Proc. Natl. Acad. Sci. USA, 74, 4370-4374. Antisenseoligodeoxynucleotides are short synthetic nucleotide sequencesformulated to be complementary to a specific gene or RNA message.Through the binding of these oligomers to a target DNA or mRNA sequence,transcription or translation of the gene can be selectively blocked andthe disease process generated by that gene can be halted. Thecytoplasmic location of mRNA provides a target considered to be readilyaccessible to antisense oligodeoxynucleotides entering the cell; hencemuch of the work in the field has focused on RNA as a target. Currently,the use of antisense oligodeoxynucleotides provides a useful tool forexploring regulation of gene expression in vitro and in tissue culture(Rothenberg, M., Johnson, G., Laughlin, C., Green, I., Craddock, J.,Sarver, N., and Cohen, J. S. (1989) J. Natl. Cancer Inst., 81:1539-1544.

SUMMARY OF THE INVENTION

The invention is related to the discovery that inhibiting the expressionof RI.sub.α in leukemia cells by contact with an antisenseO-oligonucleotides and S-oligonucleotides for RI.sub.α results in theinhibition of proliferation and the stimulation of cell differentiation.Accordingly, the invention is directed to RI.sub.α antisenseoligonucleotides and pharmaceutical compositions thereof for thetreatment of cancer.

In particular, the invention is related to 15- to 30-mer antisenseoligonucleotides which are complementary to a region in the first 100N-terminal codons of RI.sub.α (Seq. ID No:6).

The invention is also related to 15- to 30-mer antisenseoligonucleotides which are a fragment of antisense DNA complementary toRI.sub.α (Seq. ID No: 5).

The invention is also related to pharmaceutical compositions comprisingat least one 15- to 30-mer antisense oligonucleotide which iscomplementary to a region in the first 100 N-terminal codons of RI.sub.α(Seq. ID No:6); and a pharmaceutically acceptable carrier.

The invention is also related to a method for treating cancer bysuppressing growth of cancer cells susceptible to growth suppression andfor inducing cancer cell differentiation in an animal comprisingadministering to an animal in need of such treatment a cancer cellgrowth suppressing amount of an RI.sub.α antisense oligonucleotide.

DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B depict graphs showing the effect of RI.sub.α antisenseoligodeoxynucleotide (SEQ. ID NO:1) on the basal rate of growth of HL-60leukemic cells (A) and the growth of these cells when treated with cAMPanalogs or TPA (B). A, cells were grown (see the Examples) in theabsence (O) or presence () of RI.sub.α antisense oligodeoxynucleotide(15 μM). At indicated times, cell counts in duplicate were performed.Data represent the average values±SD of four experiments. B, On day 4 ofexperiment A, cells exposed or unexposed to RI.sub.α antisenseoligodeoxynucleotide (SEQ ID NO:1) were reseeded (day 0) at 5×10⁵cells/dish, and cells pre-exposed to RI.sub.α antisenseoligodeoxynucleotide were further treated with the oligomer at day 0 andday 2. cAMP analogs and TPA were added one time at day 0. Cell countswere performed on a Coulter counter on day 4. 8-Cl, 8-Cl-cAMP (10 μM);8-C1+N⁶ -B, 8-C1-cAMP (5 μM)+N⁶ -benzyl-cAMP (5 μM); TPA (10-⁸ M). Thedata represent the average values±SD of four experiments.

FIGS. 2A, 2B, 2C, 2D, 2E and 2F depict graphs showing the effect ofRI.sub.α antisense oligodeoxynucleotide (SEQ ID NO:1) on the morphologictransformation of HL-60 cells. Cells either exposed or unexposed toRI.sub.α antisense oligodeoxynucleotide were treated with cAMP analogsor TPA as described in FIG. 1B. On day 4 (see FIG. 1B), cells werewashed twice in Dulbecco's phosphate-buffered saline and were pelletedonto a glass slide by cytocentrifuge. The resulting cytopreparationswere fixed and stained by Wright's stain.×180.

FIGS. 3A and 3B depict Northern blots showing decreased RI.sub.α mRNAexpression in HL-60 leukemic cells exposed to RI.sub.α antisenseoligodeoxynucleotide (SEQ ID NO:1). Cells were either exposed orunexposed to RI.sub.α antisense oligodeoxynucleotide (15 μM) for 8 hr.Isolation of total RNA and Northern blot analysis followed the methodsdescribed in the Examples. A, ethidium bromide staining of RNA; M,markers of ribosomal RNAs; lanes 1, 2, cells unexposed or exposed toRI.sub.α antisense oligomer. B, Northern blot analysis; the samenitrocellulose filter was hybridized to both RI.sub.α and actin probesin sequential manner. Lanes 1, 2, cells unexposed or exposed to RI.sub.αantisense oligomer.

FIGS. 4A, 4B, and 4C depict SDS-PAGE showing the effect of RI.sub.αantisense oligodeoxynucleotide on the basal and induced levels ofRI.sub.α and RII.sub.β cAMP receptor proteins in HL-60 leukemic cells.Cells were either exposed to RI.sub.α antisense oligodeoxynucleotide(SEQ ID NO:1) (15 μM) or treated with cAMP analogs as described inFIG. 1. Preparation of cell extracts, the photoactivated incorporationof 8-N₃ -[³² P]cAMP and immunoprecipitation using the anti-RI.sub.α oranti-RII₆₂ antiserum and protein A Sepharose, and SDS-PAGE ofsolubilized antigen-antibody complex followed the methods described inthe Examples. Pre-immune serum controls were carried out simultaneouslyand detected no immunoprecipitated band. M, ¹⁴ C-labeled marker proteinsof known molecular weight; RI.sub.α, the 48,000 molecular weight RI(Sigma); RII.sub.α, the 56,000 molecular weight RII (Sigma). LanesRI.sub.α and RII₆₂ are from photoaffinity labeling with 8-N₃ -[³² P]cAMPonly; lanes 1 to 3, photoaffinity labeling with 8-N₃ -[³² P]cAMPfollowed by immunoprecipitation with anti-RI.sub.α or anti-RII₆₂antiserum. 8-Cl, 8-Cl-cAMP (5 μM); N⁶ -benzyl, N⁶ -benzyl-cAMP (5μM).The data in the table represent quantification by densitometric scanningof the autoradiograms. The data are expressed relative to the levels incontrol cells unexposed to RI.sub.α antisense oligomer and untreatedwith cAMP analog, which are set equal to 1 arbitrary unit. The datarepresent an average±SD of three experiments. A and B,immunoprecipitation with anti-RI.sub.α and anti-RII.sub.β antisera,respectively.

FIGS. 5A, 5B, 5C and 5D depict graphs showing the growth inhibition ofhuman cancer cell lines by RI.sub.α antisense oligodeoxynucleotidehaving SEQ ID No: 1 (O-oligo and S-oligo derivatives), compared tocontrols. Cell lines: SK-N-SH, neuroblastoma; LS-174T, colon carcinoma;MCF-7, breast carcinoma; TMK-1, gastric carcinoma. E₂, estradiol-17β.

FIGS. 6A, 6B, and 6C depict the change in morphology of SK-N-SH humanneuroblastoma cells exposed to RI.sub.α antisense oligodeoxynucleotidehaving SEQ ID No: 1.

FIGS. 7A and 7B depict graphs showing that RI.sub.α antisenseoligodeoxynucleotide and its phosphorothioate analog (SEQ ID NO:1)inhibit the in vivo growth of LS-174T human colon carcinoma in athymicmice. FIG. 7A shows the oligodeoxynucleotide concentration-dependentinhibition of tumor growth. O-oligo, RI.sub.α antisenseoligodeoxynucleotide; S-oligo, phosphorothioate analog of RI.sub.αantisense oligomer. The cholesterol pellets (total weight 20 mg)containing the indicated doses of O-oligo or S-oligo were implanted s.c.one time, at zero time, and tumor sizes were measured. Tumor volume (seeMaterials and Methods, Example 3 ) represents an average±S.D. of 7tumors. FIG. 7B shows the temporal effect of antisenseoligodeoxynucleotide phosphorothioate analogs on tumor growth. S-oligosas indicated at 0.3 mg dose in cholesterol pellets (total weight 20 mg)were implanted s.c. 2×/week, and tumor volume (see Materials andMethods, Example 3 ) represents an average±S.D. of 7 tumors.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Antisense therapy is the administration of exogenous oligonucleotideswhich bind to a target polynucleotide located within the cells. The term"antisense" refers to the fact that such oligonucleotides arecomplementary to their intracellular targets, e.g., RI.sub.α. See forexample, Jack Cohen, OLIGODEOXYNUCLEOTIDES, Antisense Inhibitors of GeneExpression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The RI.sub.αantisense oligonucleotides of the present invention include derivativessuch as S-oligonucleotides (phosphorothioate derivatives or S-oligos,see, Jack Cohen, supra) which exhibit enhanced cancer cell growthinhibitory action (see FIGS. 5 and 7A).

S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of anoligonucleotide (O-oligo) in which a nonbridging oxygen atom of thephosphate group is replaced by a sulfur atom. The S-oligos of thepresent invention may be prepared by treatment of the correspondingO-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide which is a sulfurtransfer reagent. See Iyer, R. P. et al., J. Org. Chem. 55:4693-4698(1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990),the disclosures of which are fully incorporated by reference herein.

The RI₆₀ antisense oligonucleotides of the present invention may be RNAor DNA which is complementary to and stably hybridizes with the first100 N-terminal codons of the RI.sub.α genome or the corresponding mRNA.Use of an oligonucleotide complementary to this region allows for theselective hybridization to RI.sub.α mRNA and not to mRNA specifyingother regulatory subunits of protein kinase. Preferably, the RI.sub.αantisense oligonucleotides of the present invention are a 15 to 30-merfragment of the antisense DNA molecule having SEQ ID NO:5 whichhybridizes to RI.sub.α mRNA. Alternatively, RI.sub.α antisenseoligonucleotide is a 15- to 30-mer oligonucleotide which iscomplementary to a region in the first 100 N-terminal codons of RI.sub.α(Seq. ID No:6). Most preferably, the RI.sub.α antisense oligonucleotidehas SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, or SEQ ID No:4.

Included as well in the present invention are pharmaceuticalcompositions comprising an effective amount of at least one of theRI.sub.α antisense oligonucleotides of the invention in combination witha pharmaceutically acceptable carrier. In one embodiment, a singleRI.sub.α antisense oligonucleotide is utilized. In another embodiment,two RI.sub.α antisense oligonucleotides are utilized which arecomplementary to adjacent regions of the RI.sub.α genome. Administrationof two RI.sub.α antisense oligonucleotides which are complementary toadjacent regions of the RI.sub.α genome or corresponding mRNA may allowfor more efficient inhibition of RI.sub.α genomic transcription or mRNAtranslation, resulting in more effective inhibition of cancer cellgrowth.

Preferably, the RI.sub.α antisense oligonucleotide is coadministeredwith an agent which enhances the uptake of the antisense molecule by thecells. For example, the RI.sub.α antisense oligonucleotide may becombined with a lipophilic cationic compound which may be in the form ofliposomes. The use of liposomes to introduce nucleotides into cells istaught, for example, in U.S. Pat. Nos. 4,897,355 and 4,394,448, thedisclosures of which are incorporated by reference in their entirety.See also U.S. Pat. Nos. 4,235,871, 4,231,877, 4,224,179, 4,753,788,4,673,567, 4,247,411, 4,814,270 for general methods of preparingliposomes comprising biological materials.

Alternatively, the RI.sub.α antisense oligonucleotide may be combinedwith a lipophilic carrier such as any one of a number of sterolsincluding cholesterol, cholate and deoxycholic acid. A preferred sterolis cholesterol.

In addition, the RI.sub.α antisense oligonucleotide may be conjugated toa peptide that is ingested by cells. Examples of useful peptides includepeptide hormones, antigens or antibodies, and peptide toxins. Bychoosing a peptide that is selectively taken up by the neoplastic cells,specific delivery of the antisense agent may be effected. The RI.sub.αantisense oligonucleotide may be covalently bound via the 5'OH group byformation of an activated aminoalkyl derivative. The peptide of choicemay then be covalently attached to the activated RI.sub.α antisenseoligonucleotide via an amino and sulfhydryl reactive hetero bifunctionalreagent. The latter is bound to a cysteine residue present in thepeptide. Upon exposure of cells to the RI.sub.α antisenseoligonucleotide bound to the peptide, the peptidyl antisense agent isendocytosed and the RI.sub.α antisense oligonucleotide binds to thetarget RI.sub.α mRNA to inhibit translation. See PCT ApplicationPublication No. PCT/US89/02363.

As antineoplastic agents, the RI.sub.α antisense oligonucleotides of thepresent invention are useful in treating a variety of cancers,including, but not limited to, gastric, pancreatic, lung, breast, anal,colorectal, head and neck neoplasms, neuroblastomas, melanoma andvarious leukemias.

The RI.sub.α antisense oligonucleotides of the invention may also beactive against the following tumor systems: F9 teratocarcinoma, SK-N-SHneuroblastoma, TMK-1 gastric carcinoma, HL-60 promyelocytic Leukemia,Leukemia L-1210, Leukemia P388, P1534 leukemia, Friend Virus Leukemia,Leukemia L4946, Mecca lymphosarcoma, Gardner lymphosarcoma, RidgwayOsteogenic sarcoma, Sarcoma 180 (ascites), Wagner osteogenic sarcoma,Sarcoma T241, Lewis lung carcinoma, Carcinoma 755, CD8F, MCF-7 breastcarcinoma, Colon 38, LS-174T colon carcinoma, Carcinoma 1025, Ehrlichcarcinoma (ascites & solid), Krubs 2 carcinoma (ascites), Bashfordcarcinoma 63, Adenocarcinoma E 0771, B16 Melanoma, Hardin-Passeymelanoma, Giloma 26, Miyona adenocarcinoma, Walker carcinosarcoma 256,Flexner-Jobling carcinoma, Jensen sarcoma, Iglesias sarcoma, Iglesiasovarian tumor, Murphy-Sturn lymphosarcoma, Yoshida sarcoma, Dunningleukemia, Rous chicken sarcoma, and Crabb hamster sarcoma.

The RI.sub.α antisense oligonucleotides and the pharmaceuticalcompositions of the present invention may be administered by any meansthat achieve their intended purpose. For example, administration may beby parenteral, subcutaneous, intravenous, intramuscular,intra-peritoneal, or transdermal routes. The dosage administered will bedependent upon the age, health, and weight of the recipient, kind ofconcurrent treatment, if any, frequency of treatment, and the nature ofthe effect desired.

Compositions within the scope of this invention include all compositionswherein the RI.sub.α antisense oligonucleotide is contained in an amountwhich is effective to achieve inhibition of proliferation and/orstimulate differentiation of the subject cancer cells. While individualneeds vary, determination of optimal ranges of effective amounts of eachcomponent is within the skill in the art. Typically, the RI.sub.αantisense oligonucleotide may be administered to mammals, e.g. humans,at a dose of 0.005 to 1 mg/kg/day, or an equivalent amount of thepharmaceutically acceptable salt thereof, per day of the body weight ofthe mammal being treated.

In addition to administering the RI.sub.α antisense oligonucleotides asa raw chemical in solution, the RI.sub.α antisense oligonucleotides maybe administered as part of a pharmaceutical preparation containingsuitable pharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the RI.sub.α antisenseoligonucleotide into preparations which can be used pharmaceutically.

Suitable formulations for parenteral administration include aqueoussolutions of the RI.sub.α antisense oligonucleotides in water-solubleform, for example, water-soluble salts. In addition, suspensions of theactive compounds as appropriate oily injection suspensions may beadministered. Suitable lipophilic solvents or vehicles include fattyoils, for example, sesame oil, or synthetic fatty acid esters, forexample, ethyl oleate or triglycerides. Aqueous injection suspensionsmay contain substances which increase the viscosity of the suspensioninclude, for example, sodium carboxymethyl cellulose, sorbitol, and/ordextran. Optionally, the suspension may also contain stabilizers.

The antisense oligonucleotides of the present invention may be preparedaccording to any of the methods that are well known to those of ordinaryskill in the art. Preferably, the antisense oligonucleotides areprepared by solid phase synthesis. See, Goodchild, J., BioconjugateChemistry, 1:165-167 (1990), for a review of the chemical synthesis ofoligonucleotides. Alternatively, the antisense oligonucleotides can beobtained from a number of companies which specialize in the customsynthesis of oligonucleotides.

Having now generally described this invention, the same will beunderstood by reference to an example which is provided herein forpurposes of illustration only and is not intending to be limited unlessotherwise specified. The entire text of all applications, patents andpublications, if any, cited above and below are hereby incorporated byreference.

EXAMPLES Example 1 Oligodeoxynucleotides

The 21-mer oligodeoxynucleotides used in the present studies weresynthesized at Midland Certified Reagent Co. (Midland, Tex.) and had thefollowing sequences: human RI.sub.α (Sandberg, M., Tasken, K., Oyen, O.,Hansson, V. & Jahnsen, T. (1987) Biochem. Biophys. Res Commun. 149,939-945) antisense, 5'-GGC-GGT-ACT-GCC-AGA-CTC-CAT-3' (SEQ ID No:1);human RII.sub.β (Levy, F. O., Oyen, O., Sandberg, M., Tasken, K.,Eskild, W., Hansson, V. & Jahnsen, T. (1988) Mol. Endocrinol., 2,1364-1373) antisense 5'-CGC-CGG-GAT-CTC-GAT-GCT-CAT-3'; human RII.sub.α(Oyen, O., Myklebust, F., Scott, J. D., Hansson, V. & Jahnsen, T. (1989)FEBS Lett, 246, 57-64) antisense, 5'-CGG-GAT-CTG-GAT-GTG-GCT-CAT-3'; andthe random sequence oligodeoxynucleotide was made of a mixture of allfour nucleotides at every position.

Cell Growth Experiment

Cells grown in suspension culture in RPM1 1640 medium supplemented with10% heat-inactivated fetal bovine serum, penicillin (50 U/ml),streptomycin (500 μg/ml), and 1 mM glutamine (Gibco, Grand Island, N.Y.)were seeded at 5×10⁵ cells per dish. Oligodeoxynucleotides were addedafter seeding and every 48 hr thereafter. Cell counts were performed ona Coulter counter. Cells unexposed or exposed to oligodeoxynucleotidesfor 4 days were reseeded (day 0) at 5×10⁵ cells/dish, and cellspre-exposed to the oligodeoxynucleotide were further treated with theoligomer at day 0 and day 2. cAMP analogs (kindly provided by Dr. R. K.Robins, Nucleic Acid Research Institute, Costa Mesa, Calif.) or12-O-tetradecanoylphorbol-13-acetate (TPA) were added one time at day 0.Cell counts were performed on day 4.

Immunoprecipitation of RIα and RII₆₂ cAMP Receptor Proteins afterPhotoaffinity Labeling with 8-N₃ -[³² P]cAMP

Cell extracts were prepared at 0°-4° C. The cell pellets (2×10⁶ cells),after two washes with PBS, were suspended in 0.5 ml buffer Ten (0.1MNaC1, 5 mM MgC1₂, 1% Nonidet P-40, 0.5% Na deoxycholate, 2 KIU/ml bovineaprotinin, and 20 mM Tris-HCl, pH 7.4) containing proteolysis inhibitors(Tortora, G., Clair, T. & Cho-Chung, Y. S. (1990) Proc. Natl. Acad. Sci.USA 87, 705-708), vortex-mixed, passed through a 22-gauge needle 10times, allowed to stand for 30 min at 4° C., and centrifuged at 750×gfor 20 min; the resulting supernatants were used as cell lysates. Thephotoactivated incorporation of 8-N₃ -[³² P]cAMP (60.0 Ci/mmol), and theimmunoprecipitation using the anti-RI.sub.α or anti-RII.sub.β antiserum(kindly provided by Dr. S. O. Do/skeland, University of Bergen, Bergen,Norway) and protein A Sepharose and SDS-PAGE of solubilizedantigen-antibody complex followed the method previously described(Tortora, G., Clair, T. & Cho-Chung, Y. S. (1990) Proc. Natl. Acad. Sci.USA 87, 705-708; Ekanger, R., Sand, T. E., Ogreid, D., Christoffersen,T. & Do/skeland, S. O. (1985) J. Biol. Chem. 260, 3393-3401).

cAMP-Dependent Protein Kinase Assays

After two washes with Dulbecco's phosphate-buffered saline, cell pellets(2×10⁶ cells) were lysed in 0.5 ml of 20 mM Tris (pH 7.5), 0.1 mM sodiumEDTA, 1 mM dithiothreitol, 0.1 mM pepstatin, 0.1 mM antipain, 0.1 mMchymostatin, 0.2 mM leupeptin, 0.4 mg/ml aprotinin, and 0.5 mg/mlsoybean trypsin inhibitor, using 100 strokes of a Dounce homogenizer.After centrifugation (Eppendorf 5412) for 5 min, the supernatants wereadjusted to 0.7 mg protein/ml and assayed (Uhler, M. D. & McKnight, G.S. (1987) J. Biol. Chem. 262, 15202-15207) immediately. Assays (40 μltotal volume) were performed for 10 min at 30° C. and contained 200 μMATP, 2.7×10⁶ cpm γ[³² P]ATP, 20 mM MgC1₂, 100 μM Kemptide (Sigma K-1127)(Kemp, B. E., Graves, D. J., Benjamin, E. & Krebs, E.G. (1977) J. Biol,Chem. 252, 4888-4894), 40 mM Tris (pH 7.5), ±100 μM protein kinaseinhibitor (Sigma P-3294) (Cheng, H.-C., Van Patten, S. M., Smith, A. J.& Walsh, D. A. (1985) Biochem. J. 231, 655-661), ±8 μM cAMP and 7 μg ofcell extract. The phosphorylation of Kemptide was determined by spotting20 μl of incubation mixture on phosphocellulose filters (Whatman, P81)and washing in phosphoric acid as described (Roskoski, R. (1983) MethodsEnzymol. 99, 3-6). Radioactivity was measured by liquid scintillationusing Econofluor-2 (NEN Research Products NEF-969).

Isolation of Total RNA and Northern Blot Analysis

The cells (10⁸ washed twice with phosphate-buffered saline) were lysedin 4.2M guanidine isothiocyanate containing 25 mM sodium citrate (pH7.0), 0.5% sarcosyl (N-lauroylsarcosine Na⁺), and 0.1Mβ-mercaptoethanol, and the lysates were homogenized, and total cellularRNA was sedimented through a CsCl cushion (5.7M CsCl, 10 mM EDTA) asdescribed by Chirgwin et al. (Chirgwin, J. M., Przybyla, A. E.,MacDonald, R. Y. & Rutter, W. J. (1977) Biochemistry 18, 5284-5288).Total cellular RNA containing 20 mM 3-[N-morpholine]propane-sulfonicacid (pH 7.0), 50% formamide, and 6% formaldehyde was denatured at 65°C. for 10 min and electrophoresed through a denaturing 1.2% agarose-2.2Mformaldehyde gel. The gels were then transferred to Biotrans nylonmembranes (ICN Biomedicals) by the method of Thomas (Thomas, P. S.(1980) Proc. Natl. Acad. Sci. USA 77, 5201-5205) and hybridized to thefollowing two ³² P-labeled nick-translated CDNA probes: 1.5 kilobase(kb) cDNA clone containing the entire coding region for the humancAMP-dependent protein kinase type I regulatory subunit, RI.sub.α(Sandberg, M., Tasken, K., Oyen, O., Hansson, V. & Jahnsen, T. (1987)Biochem. Biophys. Res. Commun. 149, 939-945) (kindly provided by Dr. T.Jahnsen, Institute of Pathology, Rikshospitalet, Oslo, Norway), andhuman β actin (Oncor p7000 β actin).

RESULTS

The RI.sub.α antisense oligodeoxynucleotide at 15 μM concentration hadimmediate effects on the rate of proliferation of HL-60 cells. By 4-5days in culture, while cells unexposed to RI.sub.α antisense oligomerdemonstrated an exponential rate of growth, cells exposed to theantisense oligomer exhibited a reduced growth rate and eventuallystopped replicating (FIG. 1A). This inhibitory effect on cellproliferation persisted throughout the culture period. The growthinhibition was not due to cell killing; cells were over 90% viable afterexposure to RI.sub.α antisense oligomer (15 μM) for 7 days as assessedby flow cytometry using forward and side scatter. RI.sub.α sense,RII.sub.α, or RII₆₂ antisense, or a random sequence oligodeoxynucleotidehad no such growth inhibitory effect.

Cells unexposed or exposed to RI.sub.α antisense oligodeoxynucleotidefor 4 days in culture were reseeded and examined for their response totreatment with cAMP analogs or TPA. In cells unexposed to RI.sub.αantisense oligodeoxynucleotide, 8-C1-cAMP (10 μM) produced 60% growthinhibition, and 80% growth inhibition was achieved by 8-Cl-cAMP (5 μM)plus N⁶ -benzyl-cAMP (5 μM) (FIG. 1B) (Tortora, G., Tagliaferri, P.,Clair, T., Colamonici, O. Neckers, L. M., Robins, R. K. & Cho-Chung, Y.S. (1988) Blood 71, 230-233), and TPA (10⁻⁸ M) exhibited 60% growthinhibition (FIG. 1B). In contrast, cells exposed to antisenseoligodeoxynucleotide exhibited retarded growth (25% the rate of growthof cells unexposed to the antisense oligomer) and neither cAMP analogsnor TPA brought about further retardation of growth (FIG. 1B).

HL-60 cells undergo a monocytic differentiation upon treatment withsite-selective cAMP analogs. Cells either unexposed or exposed toRI.sub.α antisense oligodeoxynucleotide were examined for theirmorphology before and after treatment with cAMP analogs. As shown inFIG. 2, in cells unexposed to RI.sub.α antisense oligomer, 8-Cl-cAMPplus N⁶ -benzyl-cAMP induced a monocytic morphologic changecharacterized by a decrease in nuclear-to-cytoplasm ratio, abundantruffled and vacuolated cytoplasm, and loss of nucleoli. Strikingly, thesame morphologic change was induced when cells were exposed to RI.sub.αantisense oligodeoxynucleotide (FIG. 2). Moreover, the morphologicchanges induced by antisense oligomer were indistinguishable from thatinduced by TPA (FIG. 2).

To provide more evidence that the growth inhibition and monocyticdifferentiation induced in HL-60 cells exposed to the RI.sub.α antisenseoligodeoxynucleotide were due to an intracellular effect of theoligomer, the RI.sub.α mRNA level was determined. As shown in FIG. 3,3.0 kb RI.sub.α mRNA (Sandberg, M., Tasken, K., Oyen, O., Hansson, V. &Jahnsen, T. (1987) Biochem. Biophys. Res. Commun. 149, 939-945) wasvirtually undetectable in cells exposed for 8 hr to RI.sub.α antisenseoligodeoxynucleotide (FIG. 3B, lane 2), and the decrease in RI.sub.αmRNA was not due to a lower amount of total RNA as shown by the ethidiumbromide staining (compare lane 2 with lane 1 of FIG. 3A). Conversely, anenhanced level of actin mRNA was detected in cells exposed to RI.sub.αantisense oligomer (FIG. 3B). Whether the increase in actin mRNA levelrepresents changes in cytoskeletal structure is not known.

The levels of cAMP receptor proteins in these cells was then determinedby immunoprecipitation using anti-RI.sub.α and anti-RII₆₂ antisera(Tortora, G., Clair, T. & Cho-Chung, Y. S. (1990) Proc. Natl. Acad. Sci.USA 87, 705-708; Ekanger, R., Sand, T. E., Ogreid, D., Christoffersen,T. & Do/skeland, S. O. (1985) J. Biol. Chem. 260, 3393-3401) afterphotoaffinity labeling of these receptor proteins with 8-N₃ -[³² P]cAMP.In control cells, treatment with 8-C1-cAMP plus N⁶ -benzyl-cAMP broughtabout a 70% reduction in RI.sub.α with a 3-fold increase in RII.sub.β,resulting in a 10-fold increase in the ratio of RII.sub.β /RI.sub.α(FIG. 4) (Cho-Chung, Y. S. (1989) J. Natl. Cancer Inst. 81, 982-987).Exposure of these cells to RI.sub.α antisense oligodeoxynucleotide for 4days brought about marked changes in both and RI.sub.α and RII₆₂ levels;an 80% reduction in RI.sub.α with a 5-fold increase in RII.sub.βresulted in a 25-fold increase in the ratio of RII.sub.β /RI.sub.αcompared with that in control cells (FIG. 4). Since growth inhibitionand differentiation were appreciable after 3-4 days of exposure toRI.sub.α antisense oligomer, the changing levels of RI.sub.α and RII₆₂proteins appears to be an early event necessary for commitment todifferentiation.

Data in FIG. 4 showed that suppression of RI.sub.α by the antisenseoligodeoxynucleotide brought about a compensatory increase in RII₆₂level. Such coordinated expression of RI and RII without changes in theamount of C subunit has been shown previously (Hofman, F., Bechtel, P.J. & Krebs, E. G. (1977) J. Biol. Chem. 252, 1441-1447; Otten, A. D. &Mcknight, G. S. (1989) J. Biol. Chem. 264, 20255-20260). The increase inRII₆₂ may be responsible for the differentiation induced in these cellsafter exposure to RI.sub.α antisense oligodeoxynucleotide. The increasein RII.sub.β mRNA or RII₆₂ protein level has been correlated with cAMPanalog-induced differentiation in K-562 chronic myelocytic leukemiccells (Tortora, G., Clair, T., Katsaros, D., Ally, S., Colamonici, O.,Neckers, L. M., Tagliaferri, P., Jahnsen, T., Robins, R. K. & Cho-Chung,Y. S. (1989) Proc. Natl. Acad. Sci. USA 86, 2849-2852) and in erythroiddifferentiation of Friend erythrocytic leukemic cells (Schwartz, D. A. &Rubin, C. S. (1985) J. Biol. Chem. 260, 6296-6303). In a recent report(Tortora, G., Clair, T. & Cho-Chung, Y. S. (1990) Proc. Natl. Acad. Sci.USA 87, 705-708), we have provided direct evidence that RII₆₂ isessential for the cAMP-induced differentiation in HL-60 cells. HL-60cells that were exposed to RII₆₂ antisense oligodeoxynucleotide becamerefractory to treatment with cAMP analogs and continued to grow.

The essential role of RII₆₂ in differentiation of HL-60 cells wasfurther demonstrated when these cells were exposed to both RI.sub.α andRII₆₂ antisense oligodeoxynucleotides simultaneously. As shown in Table1, RI.sub.α antisense oligodeoxynucleotide (SEQ ID NO:1) induced amarked increase in the expression of monocytic surface antigens [Leu 15(Landay, A., Gartland, L. & Clement, L. T. (1983) J. Immunol. 131,2757-2761) and Leu M3 (Dimitriu-Bona, A., Burmester, G. R., Waters, S.J. & Winchester, R. J. (1983) J. Immunol. 130, 145-152)] along with adecrease in markers related to the immature myelogenous cells [My9(Talle, M. A., Rao, P. E., Westberg, E., Allegar, N., Makowski, M.,Mittler, R. S. & Goldstein, G. (1983) Cell. Immunol. 78, 83.; Todd, R.F. III, Griffin, J. D., Ritz, J., Nadler, L. M. Abrams, T. & Schlossman,S. F. (1981) Leuk. Res. 5, 491)]. These changes in surface markerexpression were abolished when cells were exposed simultaneously to bothRI.sub.α and RII.sub.β antisense oligodeoxynucleotides (Table 1). RII₆₀cAMP receptor was not detected in HL-60 cells (Cho-Chung, Y. S., Clair,T., Tagliaferri, P., Ally, S., Katsaros, D., Tortora, G., Neckers, L.,Avery, T. L., Crabtree, G. W. & Robins, R. K. (1989) Cancer Invest.7(2), 161-177), and RII₆₀ antisense oligodeoxynucleotide showed nointerference with the effects of RI.sub.α antisense oligomer (Table 1).

Cells exposed to both RI.sub.α and RII₆₂ antisense oligodeoxynucleotideswere neither growth inhibited nor differentiated regardless of cAMPanalog treatment. We interpret these results to reflect the blockage ofcAMP-dependent growth regulatory pathway. Cells under these conditionsare no longer cAMP-dependent but survive and proliferate probablythrough an alternate pathway. Thus, suppression of both RI.sub.α andRII₆₂ gene expression led to an abnormal cellular growth regulationsimilar to that in mutant cell lines (Gottesman, M. M. (1980) Cell 22,329-330), those that contain either deficient or defective regulatorysubunits of cAMP-dependent protein kinase and are no longer sensitive tocAMP stimulus.

Our results demonstrated that cAMP transduces signals for dual controls,either positive or negative, on cell proliferation, depending on theavailability of RI.sub.α or RII₆₂ receptor proteins. The RI.sub.αantisense oligodeoxynucleotide which brought about suppression ofRI.sub.α along with enhancement of RII.sub.β expression led to terminaldifferentiation of HL-60 leukemia with no sign of cytotoxicity.

It is unlikely that free C subunit increase in cells exposed to RI.sub.αantisense oligodeoxynucleotide was responsible for the differentiation,because cells exposed to RII.sub.β antisense or both RI.sub.α andRII.sub.β antisense oligodeoxynucleotides, conditions which also wouldproduce free C subunit, continued to grow and became refractory to cAMPstimulus. In order to directly verify this we measuredphosphotransferase activity in cells that are exposed or unexposed tothe antisense oligodeoxynucleotides using kemptide (Kemp, B. E., Graves,D. J., Benjamin, E. & Krebs, E. G. (1977) J. Biol. Chem. 252, 4888-4894)as a substrate in the presence and absence of a saturating concentrationof cAMP and in the presence and absence of the heat-stable proteinkinase inhibitor (Cheng, H.-C., Van Patten, S. M., Smith, A. J. & Walsh,D. A. (1985) Biochem. J. 231, 655-661). This method of assay givesaccurate determination of the relative levels of dissociated C and totalC activity. Cell extracts from untreated HL-60 cells exhibited a verylow level of dissociated C and were stimulated 36-fold by cAMP (Table2). This cAMP-stimulated activity was almost completely inhibited by theheat-stable protein kinase inhibitor (Table 2), indicating that thetotal C activity measured was cAMP-dependent protein kinase. In cellsexposed to RI.sub.α antisense, RII.sub.β antisense, or RI.sub.α andRII.sub.β antisense oligodeoxynucleotide, the free C activity was notincreased as compared to unexposed control cells, although there was asmall difference in the total cAMP-stimulated activity (Table 2). Theseresults provide direct evidence that free catalytic subunit is notresponsible for the differentiation observed in HL-60 cells.

Over expression of RI.sub.α cAMP receptor protein has also been found inthe majority of human breast and colon primary carcinomas examined(Bradbury, A. W., Miller, W. R., Clair, T., Yokozaki, H. & Cho-Chung,.Y,S. (1990) Proc. Am. Assoc. Cancer Res. 31, 172), suggesting an importantin vivo role of cAMP receptor in tumor growth as well. However, theprecise role of RI.sub.α in cell proliferation is not known at present.RI.sub.α may suppress RII.sub.β production by titrating out C subunit,or it may be a transducer of mitogenic signals leading to cellproliferation. Our results demonstrate that RI.sub.α antisenseoligodeoxynucleotide provides a useful genetic tool for studies on therole of cAMP receptor proteins in cell proliferation anddifferentiation, and contribute to a new approach in the control ofmalignancy.

                  TABLE 1                                                         ______________________________________                                        Modulation of differentiation markers in HL-                                  60 cells by RI.sub.α  antisense oligodeoxynucleotide                                     Surface Makers                                               Treatment          Leu15    LeuM3   My9                                       ______________________________________                                        Control            10       2       100                                       RI.sub.α antisense                                                                         80       98       80                                       RI.sub.α antisense + RII.sub.β antisense                                              11       2       100                                       RII.sub.β antisense                                                                         13       3       100                                       RI.sub.α antisense + RII.sub.α antisense                                             85       100      80                                       ______________________________________                                    

Surface antigen analysis was performed by flow cytometry usingmonoclonal antibodies reactive with either monocytic or myeloid cells.The monoclonal antibodies used were Leu 15, Leu M3, and My9. 2×10⁴ cellswere analyzed for each sample, and cell gating was performed usingforward and side scatter. The numbers represent % positive and representthe average values of three experiments.

                                      TABLE 2                                     __________________________________________________________________________    Protein kinase activity in HL-60 cells                                                Activity -                                                                          Relative                                                                            Activity +                                                                           Relative                                                                            Stimulation                                  Treatment                                                                             cAMP  to control                                                                          cAMP   to control                                                                          (fold)                                       __________________________________________________________________________    -PKI                                                                          Control 23.0 ± 6.6                                                                       1.0   837 ± 87                                                                          1.0   36                                           RI.sub.α antisense                                                              22.9 ± 5.4                                                                       1.0   944 ± 18                                                                          1.1   41                                           RII.sub.β antisense                                                              22.8 ± 8.1                                                                       1.0   1,028 ± 154                                                                       1.2   45                                           RI.sub.α and                                                                    24.3 ± 7.0                                                                       1.1   802 ± 36                                                                          1.0   33                                           RII.sub.β antisense                                                      +PKI                                                                          Control 17.5 ± 8.7                                                                       1.0   37.0 ± 8.4                                                                        1.0   2.1                                          RI.sub.α antisense                                                              25.0 ± 8.8                                                                       1.4   22.6 ± 8.8                                                                        0.6   0.9                                          RII.sub.β antisense                                                              24.0 ± 2.6                                                                       1.4   24.8 ± 3.9                                                                        0.7   1.0                                          RI.sub.α and                                                                    19.0 ± 5.9                                                                       1.1   19.1 ± 8.2                                                                        0.5   1.0                                          RII.sub.β antisense                                                      __________________________________________________________________________

Cells were exposed to each of 15 μM concentrations of RI.sub.α,RII.sub.β, or RI.sub.α and RII.sub.β antisense oligodeoxynucleotide for4 days as shown in FIG. 1A. The data represent an average±SD ofduplicate determinations of three identical experiments.

*Picomoles phosphate transferred to Kemptide per min/mg protein.

Example 2

Next, the RI.sub.α antisense oligonucleotide having SEQ ID NO:1 wasadministered to mice having an experimental tumor. A pellet of RI.sub.αantisense oligonucleotide (25 mg/Kg) and cholesterol (1000 mg/Kg) wasimplanted s.c. in the left flank of athymic mice which had been injectedin the right flank with LS-174T human colon cancer cells (2×10⁶ cells)suspended in phosphate-buffered saline. Tumor measurements and mouseweights were recorded on the initial day of treatment (staging day), andat the end of treatment (staging day +5). The mean tumor weight change(Δ), was based on length and width measurements in millimeters. After afew days, the tumor growth was inhibited when compared to control cells(see Table 3). No change in body weight was noted in the control andtreated animals.

                  TABLE 3                                                         ______________________________________                                        Effect of RI.sub.α antisense oligodeoxynucleo-                          tide s.c. pellet on the growth of LS-174T human                               colon carcinoma in athymic mice                                                            Initial Final                                                                 mean.sup.c                                                                            mean.sup.d                                                            tumor   tumor     %                                                           wt (mg) wt (mg)   ΔT/ΔC.sup.e                        ______________________________________                                        Treatment.sup.a                                                               s.c. pellet                                                                   implanted                                                                     Control        25        450       --                                         RI.sub.α antisense                                                                     25        230       48                                         (0.5 mg)                                                                      8-Cl cAMP (1 mg).sup.b +                                                                     34        250       51                                         N.sup.6 benzyl cAMP (1 mg)                                                    ______________________________________                                         .sup.a 20 mg pellet lyophilized consisting of indicated doses of              RI.sub.α antisense or cAMP analogs plus supplement doses of             cholesterol.                                                                  .sup.b The growth inhibitory effect of these cAMP analogs correlate with      decrease in RI.sub.α (Natl. Cancer Inst. 81 982 (1989)) and is show     here for comparison.                                                          .sup.c Mean tumor weight per group (4 mice) on staging day.                   .sup.d Mean tumor weight per group on staging day +5.                         .sup.e % of change in test tumor weight (ΔT)/change in control tumo     weight (ΔC).                                                       

In other in vitro experiments, the RI.sub.α antisense oligonucleotidehaving SEQ ID NO: 1 was added to dishes containing neuroblastoma, coloncarcinoma, breast carcinoma and gastric carcinoma cells. As shown inFIG. 5, the RI.sub.α antisense oligonucleotide having SEQ ID No: 1inhibited proliferation of all cancer cell types when compared tocontrol cells. Moreover, the RI.sub.α antisense oligonucleotide havingSEQ ID No: 1 caused differentiation of the human neuroblastoma cells(see FIG. 6).

Example 3

Next, the effect of O-oligo and S-oligo RI.sub.α antisenseoligonucleotides on the growth of LS-174T human colon carcinoma inathymic mice was compared.

Materials and Methods

We synthesized [Milligen Biosearch 8700 DNA synthesizer (Bedford,Mass.)] the 21-mer antisense oligodeoxynucleotides and theirphosphorothioate analogs complementary to the human RI.sub.α, humanRII.sub.β mRNA transcripts starting from the first codon, and mismatchedsequence (random) oligomers of identical size. The oligomers had thefollowing sequences: RI.sub.α antisense,5'-GGC-GGT-ACT-GCC-AGA-CTC-CAT-3'(SEQ ID NO:1); RII.sub.α antisense,5'-CGC-CGG-GAT-CTC-GAT-GCT-CAT-3'; and random oligo,5'-CGA-TCG-ATC-GAT-CGA-TCG-TAC-3'.

LS-174T human colon carcinoma cells (2×10⁶) were injected s.c. inathymic mice, and the antisense oligodeoxynucleotides in the form ofeither a cholesterol pellet or 50% sesame oil emulsion were administereds.c. 1 week later when mean tumor sizes usually were 25-50 mg. Tumorvolume was based on length and width measurements and calculated by theformula 4/3 πr³, where r=(length+width)/4.

Results and Discussion

FIG. 7 shows the dose- and time-dependent effect of an RI.sub.αantisense oligodeoxynucleotide (O-oligo) at 0.2 and 0.5 mg doses incholesterol pellets administered s.c. one time (at zero time); itbrought about 20 and 46% growth inhibition, respectively, in 7 days whencompared with control (untreated) tumors (FIG. 7A). Strikingly, theRI.sub.α antisense phosphorothioate analog (S-oligo) at a 0.2 mg dose(cholesterol pellet, s.c.) gave a 60% growth inhibition at day 7,exhibiting a 3-fold greater potency than the O-oligo antisense (FIG.7A). The growth inhibitory effect of RI.sub.α antisense S-oligo was evengreater when animals were treated for a longer period. The RI.sub.αantisense S-oligo at a 0.3 mg dose in a cholesterol pellet, 2 times/weeks.c. implantation for 3 weeks, resulted in a 80% growth inhibition; thetumor growth almost stopped after 2 weeks of treatment (FIG. 7B).RI.sub.α antisense O-oligo or S-oligo administered s.c. as 50% sesameoil emulsion gave similar results. RI.sub.α antisense S-oligo broughtabout no apparent toxicity in animals; no body weight loss or othertoxic symptoms were observed during the 3 weeks of treatment.

The growth inhibitory effect brought about by RI.sub.α antisense S-oligowas the specific effect of the oligomer: RII₆₂ antisense or random(mismatched sequence) S-oligos of the identical size as the RI.sub.αantisense oligomer had no effect on the tumor growth (FIG. 7B).

To provide more evidence that the growth inhibition observed in coloncarcinomas in athymic mice treated with RI.sub.α antisenseoligodeoxynucleotide was due to an intracellular effect of the oligomer,the levels of RI.sub.α and RII₆₂ cAMP receptor proteins in these tumorswere determined. RI.sub.α levels were determined by immunoblotting(Ally, S., Proc. Natl. Acad. Sci. USA 85:6319-6322 (1988)) usingmonoclonal antibody against human RI.sub.α (kindly provided by Drs. T.Lea, University of Oslo, Oslo, Norway, and S. O. Do/skeland, Universityof Bergen, Bergen, Norway), and RII₆₂ was measured byimmunoprecipitation (Tortora, G., et al., Proc. Natl. Acad. Sci. USA87:705-708 (1990)) with anti-RII₆₂ antiserum (kindly provided by Dr. S.O. Do/skeland) after photoaffinity labeling of RII₆₂ with [³² P] 8-N₃-cAMP. As shown in Table 4, RI.sub.α antisense S-oligomer treatmentbrought about a marked reduction (80% decrease) of RI.sub.α level intumors as compared with that in untreated control tumors. Thissuppression of RI.sub.α expression by RI.sub.α antisense S-oligomerbrought about a 2-fold increase in RII₆₂ level (Table 4). Suchcoordinated expression of RI.sub.α and RII.sub.β without changes in theamount of catalytic subunit of protein kinase has been shown in HL-60leukemia cells that demonstrated growth inhibition and differentiationupon exposure to RI.sub.α antisense oligodeoxynucleotide. On the otherhand, a 50% increase in RI.sub.α level along with 80% suppression inRII.sub.β level was observed in tumors after treatment with RII.sub.βantisense S-oligomer (Table 4) which had no effect on tumor growth (FIG.7). Random (mismatched sequence) S-oligomer which had no effect on tumorgrowth (FIG. 7) also showed no effect on RI.sub.α levels (Table 4).Thus, reduction in RI.sub.α expression appears to trigger a decrease orhalt in tumor growth upon treatment with RI.sub.α antisense oligomer.Our results demonstrated that cAMP transduces signals for dual control,either positive or negative, on cell proliferation, depending on theavailability of RI.sub.α or RII.sub.β receptor proteins. The RI.sub.αantisense oligodeoxynucleotide, which suppressed RI.sub.α and enhancedRII₆₂ expression, led to inhibition of in vivo growth of solid coloncarcinoma in athymic mice with no symptoms of toxicity in animals. Thephosphorothioate analog (S-oligo) of RI.sub.α antisense oligomerexhibited a greater potency than the antisense of unmodifiedoligodeoxynucleotide (O-oligo). It has been shown that S-oligos, ascompared with O-oligos, more readily enter cells, are more resistant toendonucleases, and yet exhibit high efficacy in hybridization withtarget mRNAs or DNAs (Stein, C. A., et al., In: J. S. Cohen (ed.),Oligodeoxynucleotides: Antisense Inhibitors of Gene Expression, pp.97-117. Boca Raton, Fla., CRC Press, Inc. (1989)).

These results demonstrate here for the first time the striking in vivoeffect of antisense oligodeoxynucleotide in the suppression ofmalignancy. The depletion of RI.sub.α, the type I regulatory subunit ofcAMP-dependent protein kinase, by means of an antisenseoligodeoxynucleotide, especially with its phosphorothioate analog, leadsto a successful halt of tumor growth in vivo with no symptoms oftoxicity, suggesting great potential of this antisenseoligodeoxynucleotide for clinical application.

                  TABLE 4                                                         ______________________________________                                        Suppression of RI.sub.α cAMP Receptor Expression by RI.sub.α      Antisense Oligodeoxynucleotide (S-oligo) Results in                           Compensatory Increase in RII.sub.β Receptor                                           Relative Levels                                                  Treatment      RI.sub.α                                                                          RII.sub.β                                       ______________________________________                                        None           1.0 ± 0.1                                                                            1.0 ± 0.1                                         RI.sub.α antisense                                                                      0.2 ± 0.03                                                                          2.0 ± 0.2                                         S-oligo                                                                       RII.sub.β antisense                                                                     1.5 ± 0.2                                                                             0.2 ± 0.02                                       S-oligo                                                                       Random S-oligo 1.0 ± 0.1                                                                            1.0 ± 0.1                                         ______________________________________                                    

Treatment with S-oligos as indicated were the same as that in FIG. 7B.At the end of the experiment (3 weeks), tumor extracts were prepared aspreviously described (Ally, S. et al., Cancer Res. 49:5650-5655 (1980))and immunoblotting and immunoprecipitation of RI.sub.α and RII.sub.β,respectively, were performed as previously described by Ally, S., etal., Proc. Natl. Acad. Sci. USA 85:6319-6322 (1988) and Tortora, G., etal., Proc. Natl. Acad. Sci. USA 87:705-708 (1990). Data are fromquantification by densitometric scanning of autoradiograms. Data areexpressed relative to levels in control tumors (no treatment), which areset to equal to one as an arbitrary unit.

Data represent an average±S.D. of 7 tumors.

In the following sequence listing, Seq ID No: 1 represents an antisensesequence corresponding to the first 7 N-terminal codons for RI.sub.α.Seq ID No: 2 represents an antisense sequence corresponding to the8^(th) -13^(th) codon for RI.sub.α. Seq ID No: 3 represents an antisensesequence corresponding to the 14^(th) -20^(th) codon for RI.sub.α. SeqID No: 4 represents an antisense sequence corresponding to the 94^(th)-100^(th) codon for RI.sub.α. Seq ID No: 5 represents an antisensesequence corresponding to the 1^(st) -100^(th) codon for RI.sub.α. SeqID No: 6 represents the sense sequence corresponding to the 1^(st)-100^(th) codon for RI.sub.α.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 7                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iv) ANTI-SENSE: yes                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGCGGTACTGCCAGACTCCAT21                                                       (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 bases                                                          (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iv) ANTI-SENSE: yes                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GCGTGCCTCCTCACTGGC18                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iv) ANTI-SENSE: yes                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GAGCTCACATTCTCGAAGGCT21                                                       (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iv) ANTI-SENSE: yes                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GATAGCACCTCGTCGCCTCCT21                                                       (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 300 bases                                                         (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iv) ANTI-SENSE: yes                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GATAGCACCTCGTCGCCTCCTACCTTTAACCACTGGGTTGGGTGGAGGAGGAGAAATCTC60                ATCCTCCCTTGAGTCTGTACGAGTGCCTGCTTTCTGCAGATTGTGAATCTGTTTTGCCTC120               CTCCTTCTCCAACCTCTCAAAGTATTCCCTGAGGAATGCCATGGGACTCTCAGGTCGAGC180               AGTGCACAACTGCACAATAGAATCTTTGAGCAGTGCTTGAATGTTATGCTTCTGGACGTA240               GAGCTCACATTCTCGAAGGCTGCGTGCCTCCTCACTGGCGGCGGTACTGCCAGACTCCAT300               (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 300 bases                                                         (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iv) ANTI-SENSE: no                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       ATGGAGTCTGGCAGTACCGCCGCCAGTGAGGAGGCACGCAGCCTTCGAGAATGTGAGCTC60                TACGTCCAGAAGCATAACATTCAAGCACTGCTCAAAGATTCTATTGTGCAGTTGTGCACT120               GCTCGACCTGAGAGACCCATGGCATTCCTCAGGGAATACTTTGAGAGGTTGGAGAAGGAG180               GAGGCAAAACAGATTCAGAATCTGCAGAAAGCAGGCACTCGTACAGACTCAAGGGAGGAT240               GAGATTTCTCCTCCTCCACCCAACCCAGTGGTTAAAGGTAGGAGGCGACGAGGTGCTATC300               (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: Nucleic acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (iv) ANTI-SENSE: No                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CGATCGATCGATCGATCGTAC21                                                       __________________________________________________________________________

I claim:
 1. A method for reducing growth of cancer cells susceptible togrowth reduction in a mammal comprising administering subcutaneously orparenterally to a mammal in need of such treatment a cancer cellgrowth-reducing amount of at least one 15- to 30-mer antisense 5' to3'-oligonucleotide which is complementary to a region in the nucleicacid encoding the first 100 N-terminal amino acids of RI.sub.α, whichnucleic acid consists of the nucleotide sequence shown as SEQ. ID No:6.2. The method of claim 1, wherein said antisense 5' to3'-oligonucleotide is a 5' to 3'-oligodeoxynucleotide.
 3. The method ofclaim 1, wherein said antisense 5' to 3'-oligonucleotide is a 21-merconsisting of the nucleotide sequence shown as SEQ. ID No:1.
 4. Themethod of claim 1, wherein said antisense 5' to 3'-oligonucleotide is an18-mer consisting of the nucleotide sequence shown as SEQ. ID No:2. 5.The method of claim 1, wherein said antisense 5' to 3'-oligonucleotideis a 21-mer consisting of the nucleotide sequence shown as SEQ. ID No:3.6. The method of claim 1, wherein said antisense 5' to3'-oligonucleotide is a 21-mer consisting of the nucleotide sequenceshown as SEQ. ID No:4.
 7. The method of claim 1, wherein said cancer isselected from the group consisting of leukemia, melanoma, sarcoma, andcarcinoma.
 8. The method of claim 1, wherein said antisense 5' to3'-oligonucleotide is administered as part of a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier.
 9. Themethod of claim 1, wherein said antisense 5' to 3'-oligonucleotide is a5' to 3' O-oligodeoxynucleotide.
 10. The method of claim 1, wherein saidantisense 5' to 3 '-oligonucleotide is a 5' to3'-oligodeoxynucleotide-phosphorothioate.